This invention relates to the adsorbent for urokinase and the production of highly pure urokinase from human urine by affinity chromatography on the adsorbent.
Urokinase is a plasminogen-activating enzyme found in trace amount in human urine and is used as an effective thrombolytic agent and a drug used together with anticancer. High purity of urokinase is required because these drugs are used by an intravenous injection.
Recently, affinity chromatography began to be used as a method for the purification of human urokinase. For example, the method using basic amino acid, i.e. lysine, arginine, as a ligand [Japanese Patent Publication No. 441953/1976, Japanese Patent Publication No. 20596/1976, Japanese Patent Publication (Kokai Koho) No. 95183/1976, Japanese Patent Publication No. (Kokai Koho) 35481-35483/1976), and the method using an urokinase inhibitor contained in the tissue of the placenta as a ligand [Japanese Patent Publication No. 205977/1976] are known. It is, however, difficult to obtain a large amount of those materials used as a ligand in those methods. Furthermore, those affinity materials have not enough affinity to adsorb urokinase specifically from urokinase solution and the washing solution must be low in the concentration of salt contained therein when washing the affinity column before the elution of urokinase therefrom, which means that it is difficult to accomplish satisfactory purification of urokinase by those known methods.
Further, an adsorbent for urokinase similar to the adsorbent of the invention is known (Lass Holmberg et al., Biochimica et Biophysica Acta 445, 215, 1976). In the adsorbent, p-aminobenzamidine is coupled to agarose through a 6-carbon spacer. However, also these adsorbents have not enough affinity to urokinase in a condition of high salt concentration, therefore, using a high salt concentration of crude urokinase as a raw material causes leakage of urokinase and a high salt concentration of washing solution can not be used to attain efficient purification of urokinase.